ABSTRACT Glioblastoma (GBM) is the most common primary brain tumor with a median survival of only 12-15 months despite advanced therapies. These disappointing clinical outcomes indicate that more efforts are required to better understand GBM pathogenesis in order to provide the foundation for identifying new effective approaches to target GBM. Research in our laboratories has focused on understanding the underlying mechanisms driving metabolism alterations in GBM. We demonstrated that SREBP-1, a master transcription factor regulating lipogenesis, is highly upregulated in GBM by oncogenic EGFR/PI3K/Akt signaling. More recently, we found a novel molecular connection between the hexosamine biosynthesis pathway (HBP) and SREBPs. We demonstrated that glucose, through the HBP pathway, increases the N-glycosylation of SCAP, the key transporter for SREBPs, promoting SREBP activation and lipid synthesis. However, the mechanisms regulating the HBP-N-glycosylation pathway in GBM and other malignancies are poorly understood. Moreover, the importance of this pathway in GBM growth is also unknown. HBP is regulated by the GFAT1, GNPNAT1, PGM3 and UAP1 enzymes that sequentially convert fructose-6-phosphate to UDP-GlcNAc, the key metabolite for initial synthesis of N-glycan, which is regulated by the DPAGT1 enzyme. To explore how the HBP- N-glycosylation pathway is regulated in GBM, we performed multiple preliminary experiments showing that: 1) all the enzymes above are highly expressed in tumor tissues from GBM patients; 2) EGFRvIII, a constitutively active EGFR mutant, significantly upregulates the expression of these enzymes in GBM cells; 3) pharmacological inhibition of SREBP-1 markedly reduces the protein levels of all enzymes, while only downregulating the mRNA levels of GFAT1, PGM3 and UAP1; 4) removing N-glycan binding using PNGase significantly increased the mobility of GNPNAT1 and DPAGT1 on SDS-PAGE, indicating that both enzymes are modulated by N- glycosylation. Based on these preliminary data, we hypothesize that SREBP-1, activated by oncogenic EGFR signaling, transcriptionally upregulates GFAT1, PGM3 and UAP1 expression to enhance hexosamine synthesis, which in turn promotes the N-glycosylation of GNPNAT1, DPAGT1 and SCAP to increase their stability and activity, leading to a feedforward loop enhancing HBP-N-glycosylation and lipogenesis to promote GBM growth. In this study, we will determine the transcriptional regulation of GFAT1, PGM3 and UAP1 by EGFR-SREBP-1 signaling, and delineate the regulation and role of the N-glycosylation on GNPNAT1 and DPAGT1 proteins in GBM (Aim1). We will further elucidate the importance of HBP-N-glycosylation on GBM growth by examining the effects of genetic inhibition of GFAT1 or mutation of GNPNAT1 or DPAGT1 N-glycan binding sites on tumor growth (Aim 2). Completion of this study will significantly improve our understanding of GBM biology and provide insights for the development of potential new strategy to combat this deadly cancer.